title: A conserved aromatic residue regulating photosensitivity in short-wavelength sensitive cone visual pigments.
abstract: Visual pigments have a conserved phenylalanine in transmembrane helix 5 located near the β-ionone ring of the retinal chromophore. Site-directed mutants of this residue (F207) in a short-wavelength sensitive visual pigment (VCOP) were studied using UV-visible spectroscopy to investigate its role in photosensitivity and formation of the light-activated state. The side chain is important for pigment formation: VCOP(F207A), VCOP(F207L), VCOP(F207M), and VCOP(F207W) substitutions all bound 11-cis-retinal and formed a stable visual pigment, while VCOP(F207V), VCOP(F207S), VCOP(F207T), and VCOP(F207Y) substitutions do not. The extinction coefficients of all pigments are close, ranging between 35800 and 45600 M⁻¹ cm⁻¹. Remarkably, the mutants exhibit an up to 5-fold reduction in photosensitivity and also abnormal photobleaching behavior. One mutant, VCOP(F207A), forms an isomeric composition of the retinal chromophore after illumination comparable to that of wild-type VCOP yet does not release the all-trans-retinal chromophore. These findings suggest that the conserved F207 residue is important for a normal photoactivation pathway, formation of the active conformation and the exit of all-trans-retinal from the chromophore-binding pocket.
authors.affiliation: Departments of Neuroscience and Physiology, Biochemistry and Molecular Biology, and Ophthalmology, SUNY Upstate Medical University, Syracuse, New York 13210, United States.
authors.forename: Colleen M
authors.initials: CM
authors.lastname: Kuemmel
authors.forename: Megan N
authors.initials: MN
authors.lastname: Sandberg
authors.forename: Robert R
authors.initials: RR
authors.lastname: Birge
authors.forename: Barry E
authors.initials: BE
authors.lastname: Knox
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D055358:Cone Opsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry*; D038761:Photobleaching; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012172:Retinaldehyde / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D014981:Xenopus; D029867:Xenopus Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D055358:Cone Opsins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D010649:Phenylalanine; D012172:Retinaldehyde